The goal of this project is to examine the role of the p50 subunit of NF-?B in the response to DNA alkylation damage as a prerequisite to improving the clinical management of malignant glioma. Malignant glioma affects over 20,000 new patients in the US each year and despite aggressive therapy, patient survival remains about 1 year. The alkylating agent, temozolomide (TMZ), is now the standard chemotherapeutic used in the treatment of malignant glioma, and while it improves overall survival, many patients experience minimal benefit. Although the upstream lesions and repair mechanisms that modulate cytotoxicity by TMZ are well elucidated, the downstream signaling that ultimately mediates cell death remains less well studied. Importantly, downstream pathways represent targets that can be modulated to improve the therapeutic effect. The central hypothesis of our work is that NF-?B, and p50 signaling in particular, regulates the cytotoxic response to TMZ and that therapeutic modulation of this pathway can improve the effectiveness of TMZ. Our aims examine sequential aspects of the p50 response to uncover novel strategies to improve the treatment of this disease. In Aim 1, we will extend our studies of the p50 subunit itself, and its post-translational modification (PTM), to examine the hypothesis that phosphorylation-dependent p50 mono-ubiquitination is necessary for cytotoxicity by TMZ. Using unbiased analysis, we have identified the tumor suppressor, Bard1, as being the E3-ligase that mediates p50 mono-ubiquitination. In Aim 2, we shift our analysis to focus on the p50 half of the NF-?B consensus element (?B-site). Specifically, we have identified the RNA helicase, UAP56, as a novel interacting partner of the ?B-site that is required for cytotoxicity by TMZ. We will examine the hypothesis that promoter specific sumoylation of UAP56 in response to TMZ transcriptionally regulates the NF-?B response to promote cytotoxicity. Finally, in Aim 3, we will translate our studies to examine clinical malignant gliomas based on the preliminary observation that the p50 co-regulator, Bcl3, is necessary for induction of p50-dependent anti- apoptotic factors in response to TMZ. Specifically, we will test the hypothesis that Bcl3 expression is a clinical biomarker in malignant glioma and that targeting Bcl3-dependent factors enhances the overall anti-glioma effect. Crucially, we have identified carbonic anhydrase II (CA2) as a unique Bcl3-dependent factor that can be targeted to sensitize tumors that are predicted to respond poorly to TMZ. These complimentary aims build a general picture of how p50 interacts with co-regulators, in a promoter-specific manner to mediate the response to DNA damage. In addition, from a broader perspective, given that p50 mediates the response to any agent or process that activates ATR and replication stress, the results of the current project will also expand our general understanding of how cells respond to replication stress induced not only exogenously but also during normal replication.